Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agoniststimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.Platelets play a critical role in thrombosis and hemostasis. At sites of vascular injury, platelets are activated by various soluble agonists such as thrombin and ADP and adhesive proteins such as collagen and von Willebrand factor. Although different agonists induce platelet activation via different signaling pathways, the signals induced by different agonists converge to common signaling events such as calcium mobilization and activation of the ligand binding function of the integrin ␣ IIb ␤ 3 that mediates platelet aggregation (1, 2). An important feature of platelet activation is the ability to self-amplify the signals, which allows low concentrations of agonists to induce maximal platelet responses. This feature is particularly important in arteries where fast flow of blood may quickly dilute soluble agonists. One important mechanism of selfamplification is the secretion of platelet granule contents such as platelet agonists ADP and serotonin and adhesive proteins von Willebrand factor and fibrinogen (3). The secreted platelet agonists and adhesive proteins, via various pathways, form "positive feedback loops" that greatly amplify and stabilize platelet aggregation, thus sensitizing platelets to low doses of platelet agonists. The signaling mechanism leading to platelet granule secretion is not totally understood. We have recently shown that nitric oxide (NO) 3 synthesized by NO synthase 3 (NOS3, also called eNOS) stimulates soluble guanylyl cyclase and induces cGMP elevation and activation of cGMP-dependent protein kinase (PKG), leading to secretion of platelet granules and the second wave of pla...

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.Development of thrombotic diseases involves the injury or dysfunction of the blood vessel wall and activation of blood platelets (1). Upon exposure to agonists such as thrombin, ADP, collagen, and von Willebrand factor (VWF), 2 platelets become "activated" and aggregate to form primary thrombi (1, 2). Activated platelets secrete large quantities of ADP, serotonin, and other factors that amplify platelet activation and stabilize platelet aggregates (3). Activated platelets also secrete pro-coagulation, pro-inflammatory, and growth factors (3-5). Thus, platelet activation plays a major role not only in acute arterial thrombosis but also in the development of chronic vascular diseases, such as atherosclerosis, which in turn causes thrombosis (1, 6).A major advance in the field of vascular biology in the last century was the discovery of the vessel dilator, nitric oxide (NO) (7-9). NO is a short-lived messenger molecule synthesized from L-arginine by a family of enzymes known as nitric-oxide synthases (NOS). Three isoforms of NOS enzymes are known (10 -12): NOS1 (neuronal NOS), NOS2 (inducible NOS), and NOS3 (endothelial NOS). NOS3 is the major isoform known to be expressed in platelets (13). One of the major functions of NO is to stimulate soluble guanylyl cyclase (sGC) and increase the synthesis of cyclic guanosine monophosphate (cGMP) that serves as a secondary messenger regulating the function of cGMP-dependent protein kinase (PKG), cGMP-dependent ion channels, and cGMP-regulated phosphodiesterases (7). High concentrations of NO can also chemically modify (nitrosylation and nitration) proteins and, thus, affect cell functions in a cGMP-independent manner (7, 14 -16). NO is involved in diverse processes, such as smooth muscle relaxation, neurotransmission, immune responses, and inflammation (7). It has been a prevailing concept that NO, by elevating intracellular cGMP, inhibits platelet activation (8). This concept is supported by data that high concentrations of NO donor compounds inhibit platelet activation (17-19). However, the concept...

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNAdamaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.acetylated p53 ͉ inositol signaling ͉ nuclear translocation I nositol lipids participate in a variety of intracellular signaling pathways including cytoskeletal dynamics, intracellular membrane trafficking, cell proliferation, and apoptosis (1, 2). In response to agonists, the phosphoinositide profile is modulated by phospholipases, lipid kinases, and lipid phosphatases. The lipid messengers transduce signals through binding to proteins with binding domains specific for different phosphoinositides.The most recently discovered of the seven known phosphoinositides is phosphatidylinositol 5-phosphate (PtdIns-5-P), and its function is the least understood (3). The origin of PtdIns-5-P in cells was until recently unknown. A study of changes in the cellular levels of PtdIns-5-P suggested that PtdIns-5-P arises from the action of a phosphatase rather than a kinase (4). Our discovery of two phosphatases that convert PtdIns-4,5-P 2 to PtdIns-5-P provides a route for synthesis of this lipid (5). Recently, it was suggested that PtdIns-5-P specifically interacts with a plant homeodomain (PHD) finger of inhibitor of growth protein-2 (ING2) protein, and that this interaction is required for ING2-dependent activation of p53, which leads to increased apoptosis (6). This suggestion was based on the finding that RNAi of ING2 or overexpression of the phosphatidylinositol phosphate kinase (PIPK) type II␤, an enzyme that converts PtdIns-5-P to PtdIns-4,5-P 2 , decreases apoptosis. Thus, it was presumed that both ING2 and PtdIns-5-P were required for acetylation of p53, although cellular PtdIns-5-P was not measured in that study (6).The ING2 is a member of the inhibitor of growth family and acts as a cofactor on the histone acetyltransferase complex that functions in chromatin remodeling and p53 acetylation and activation (7). Mutation of the PHD finger that renders PtdIns-5-P-binding defective...